Method of screening cell death modulators

ABSTRACT

The invention provides methods and compositions for identifying agents which modulate cell death, indicated e.g. by the expression of caspase-2 and/or caspase-7, in GDNF family growth factor deprived neuronal or nonneuronal cells. The methods for identifying such agents find particular application in drug development.

The invention provides methods and compositions for identifying agentswhich prevent apoptosis, i.e. cell death, in growth factor deprivedneuronal or nonneuronal cells.

BACKGROUND OF THE INVENTION

Programmed cell death is a process by which unwanted cells areintentionally removed, due to either physiological or pathologicalreasons. Morphological appearance of the dying cells and the deathprogram (molecular and cellular death pathways) can differ remarkablybetween cell types and death stimuli (Clarke, 1990; Zimmermann et al.,2001; Leist and Jäättelä, 2001). Currently, two death pathways have beendescribed in detail: the death receptor (extrinsic) and mitochondrial(intrinsic) pathway. The extrinsic pathway is activated by tumournecrosis factor receptor superfamily death receptor ligation (Vincenz,2001). The death-inducing signaling complex, assembled directly at thedeath receptors, activates the initiator caspase-8 that in turnactivates caspases-3, -6 and -7. Activation of the intrinsic deathpathway leads to release of cytochrome c (but also other apoptoticmolecules) from the mitochondrial intermembrane space to the cytosol.Cytosolic cytochrome c triggers formation of the apoptosome thatactivates the initiator caspase-9 followed by activation of caspase-3,-6 and -7. It was shown recently that caspase-2 is activated upstream ofmitochondria and may participate in the activation ofmitochondria-related death events (Guo et al., 2002; Lassus et al.,2002; Read et al., 2002).

Mitochondrial death pathway is triggered by different modes of cellularstress and in some cells by removal (deprivation) of survival (trophic)factors. The well-characterized example of such cells is the neonatalmouse or rat sympathetic neurons that critically depend on nerve growthfactor (NGF) for survival. Withdrawal of NGF from the culturedsympathetic neurons leads to the following events. The protein levelsand phosphorylation of transcription factor c-Jun are increased (Estuset al., 1994; Ham et al., 1995; Virdee et al., 1997; Eilers et al.,1998), pro-apoptotic protein Bax is translocated from the cytosol to themitochondria Deckwerth et al., 1996; Putcha et al., 1999), cytochrome cis released from the mitochondria to the cytosol (Deshmukh and Johnson,1998; Neame et al., 1998; Martinou et al., 1999) together withSmac/DIABLO, a protein that releases caspases from the Inhibitor ofApoptosis Proteins (Deshmukh et al., 2002). As a result, caspase-9,caspase-3 (Deshmukh et al., 2000; Deshmukh et al., 2002) but alsocaspase-2 (Troy et al., 2001) are activated. All these events arecritically required for the NGF deprivation-induced death. The neuronsthen exhibit classical features of apoptosis, including condensation ofchromatin, cleavage of DNA but also increased autophagy (Martin et al.,1988; Pittman et al., 1993; Edwards and Tolkovsky, 1994; Xue et al.,1999) and die finally in the culture by secondary necrosis.

In addition to these two, several other death pathways exist (Clarke,1990; Leist and Jäättelä, 2001) but these remain largely unknown. Cellsin which the intrinsic apoptotic pathway is blocked can still be inducedto die, both in vitro and in vivo, often with nonapoptoticultrastructure (Yaginuma et al., 2001; Oppenheim et al., 2001; Zaidi etal., 2001; Marsden et al., 2002). Recently, novel death pathways havebeen proposed for the dependence receptors that trigger death by a novelmechanism, when not occupied with their cognate ligands, whereasligation of the receptors blocks death (Rabizadeh et al., 1993; Ellerbyet al., 1999; Bordeaux et al., 2000; Llambi et al., 2001; Thibert etal., 2003). In the case of the Deleted in Colorectal Cancer receptor,this mechanism includes direct interaction of caspases with the receptorand does not require the death receptors or mitochondrial pathways.Certainly further death pathways exist.

NGF is currently the best-characterized neurotrophic factor. Althoughmany more neurotrophic factors are known that promote survival ofdifferent types of neurons (Huang and Reichardt, 2001), the deathpathways activated by their withdrawal are virtually unstudied. Glialcell line-derived neurotrophic factor (GDNF) (Airaksinen and Saarma,2002) is a neurotrophic factor that promotes survival of severalneuronal populations, including neonatal rat sympathetic neurons(Kotzbauer et al., 1996). NGF and GDNF signal via different receptorsystems: TrkA/p75 for NGF and Ret/GFRα1 complex for GDNF. We comparedthe death programs triggered in the same cell type (sympathetic neurons)by removal of two different neurotrophic factors (NGF or GDNF).Surprisingly we found that the death pathways activated in these twocases differ considerably.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for identifying agentswhich modulate cell death, indicated e.g. by the expression of caspase-2and/or caspase-7, in GDNF family growth factor deprived neuronal ornonneuronal cells. The methods for identifying such agents findparticular application in commercial drug screens.

In particular, the invention provides a method of screening cell deathmodulators of neuronal or nonneuronal cells in a GDNF family growthfactor dependent cell culture system comprising the steps of removingthe GDNF family growth factor from the culture system, introducing acandidate modulator agent into the culture system, and determining theactivity of caspase-2 and/or caspase-7.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C. Culture conditions for GDNF-responsive newborn ratsympathetic neurons from the superior cervical ganglion. (1A) Newlyisolated neurons were grown with GDNF (100 ng/ml), NGF (30 ng/ml) orwithout neurotrophic factors for eight days. The living neurons werecounted daily and expressed as percent of initial neurons, counted twohours after plating. The mean±standard error of the mean of threeindependent cultures is shown for each data point. (1B) Newly isolatedneurons were maintained with different doses of GDNF for six days. Theliving neurons were counted daily and expressed as percent of initialneurons, counted two hours after plating. For comparison, survival withNGF (30 ng/ml) is also shown. The mean±standard error of the mean ofthree independent cultures is shown for each data point. (1C) Neuronswere first maintained with GDNF (100 ng/ml) or NGF (30 ng/ml) for sixdays. Neurotrophic factors were then removed (0-day of deprivation) andthe neurons were grown further without them. Living neurons were counteddaily and expressed as percent of initial neurons counted immediatelyafter factor deprivation. The mean±standard error of the mean of sixindependent cultures is shown for each data point.

FIGS. 2A, 2B, and 2C. Cytochrome c is not released from the mitochondriaof GDNF-deprived sympathetic neurons. (2A) Upper row, micrographs of theneurons deprived of GDNF or NGF for 48 h in the presence of caspaseinhibitor BAF, or maintained with these factors, and immunostained withcytochrome c antibodies. Corresponding phase contrast images are shownon the lower row. Note that weak and diffuse immunostaining is barelyvisible on the NGF-deprived neurons, whereas almost all GDNF-deprivedneurons stain strongly, as the neurons maintained with the factors.Scale bar: 100 μm. (2B) Upper row, typical cytochrome c immunostainingpatterns of GDNF- or NGF-deprived or -maintained neurons. Correspondingphase contrast images are shown on the lower row. Note that in spite ofpyknotic appearance, the GDNF-deprived neurons show strong punctateimmunostaining (mitochondrial localization), whereas the NGF-deprivedneurons stain weakly and diffusely (cytosolic localization). Levels ofthe images were equally enhanced with Adobe Photoshop software. Scalebar: 10 μm. (2C) Quantitation of the neurons deprived of neurotrophicfactors for 48 h with or without BAF and having punctate pattern ofcytochrome c immunostaining, calculated as percent of all neurons.Experiments with or without BAF were performed separately (fourindependent experiments for both) and combined to the same figure. Themean±standard error is shown for each data point. Statisticalsignificance of the differences between factor-maintained and -deprivedgroups was estimated by Student's t test.

FIGS. 3A and 3B. Bax is not required for death of GDNF-deprivedsympathetic neurons. (3A) GDNF- or NGF-deprived neurons weremicroinjected with expression plasmids for Ku70 or empty vector. (B)Neurons were deprived of GDNF or NGF in the presence of Ku70-derivedBax-blocking peptide V5 or control peptide I5. In (3A and 3B), livingneurons were counted 72 h later and expressed as percent of initialneurons. The mean±standard error of the mean of three (3A) or four (3B)independent experiments is shown for each data point. Statisticalsignificance of the differences was estimated by one-way ANOVA and posthoc Tukey's honestly significant difference test.

FIG. 4. Inhibition of individual caspases in GDNF- or NGF-deprivedsympathetic neurons. GDNF- or NGF-selected sympathetic neurons weremicroinjected with expression plasmids for dominant negative (DN)mutants of indicated caspases and the neurotrophic factors were thendeprived. Living neurons were counted 72 h later and expressed aspercent of initial neurons. The mean±standard error of the mean of three(four for DN caspase-9) independent cultures is shown for each datapoint. Individual caspases were studied in different experiments and arecombined to the same figure. Data of each DN-caspase were compared toaveraged vector-controls and uninjected controls by one-way ANOVA andpost hoc Tukey's honestly significant difference test.

FIGS. 5A, 5B, 5C, and 5D. Involvement of proteins of the apoptoticmachinery in the death of GDNF- or NGF-deprived sympathetic neurons.(5A) Overexpressed Bcl-x_(L) rescues NGF-deprived but not GDNF-deprivedneurons, whereas overexpressed Bax kills both types of neurons. (5B)Broad-range caspase inhibitor BAF (50 μg/ml) protects both GDNF- andNGF-deprived neurons. (5C) Overexpressed XIAP protects NGF-deprived butnot GDNF-deprived neurons. (5D) Overexpressed dominant negative FADD (DNFADD) does protect neither GDNF- nor NGF-deprived neurons. In (5A-D),the living neurons were counted 72 h after treatment and neurotrophicfactor deprivation, and expressed as percent of initial neurons. Themean±standard error of the mean of three independent cultures is shownfor each data point. Data of Bcl-x_(L)-(5A), XIAP- (5C) or DNFADD-injected (5D) neurons were compared to respective vector-injectedor untreated neurotrophic factor-deprived controls by one-way ANOVA andpost hoc Tukey's honestly significant difference test. Data ofBAF-treated neurons (5B) were compared to untreated controls byStudent's t test.

FIGS. 6A and 6B. Activation of MLK and c-Jun is required for the deathof GDNF-deprived sympathetic neurons. (6A) Quantitation of neurons withstrong nuclear immunostaining for phosphorylated c-Jun, expressed aspercent of all neurons. Neurons were deprived of neurotrophic factors inthe presence of caspase inhibitor BAF for 48 hours and immunostainedwith antibodies to phosphorylated serines 63 or 73 of c-Jun.Control-neurons maintained with GDNF or NGF were stained as well. Themean±standard error of the mean of four (for P-Ser-63) or three (forP-Ser-73) independent cultures is shown for each data point.Neurotrophic factor-maintained and -deprived groups were compared byStudent's t test. (6B) Typical examples of weak (GDNF-deprived neurons)or strong (NGF-deprived neurons) nuclear immunostaining. Correspondingphase contrast images are shown on the right column. Levels of thefluorescent images were equally enhanced with Adobe Photoshop software.Scale bar, 10 μm.

FIGS. 7A, 7B and 7C. Autophagy is greatly enhanced in GDNF-deprivedsympathetic neurons. (7A) Ultrastructure of a typical GDNF-maintainedneuron with normal mitochondria (m), Golgi complex (G), and two darkautolysosomes (a) that are sparse in these neurons. (7B) TypicalGDNF-deprived neuron from sister dish showing largely increased numberof autolysosomes, but normal mitochondria and Golgi complex. N: nucleus.(7C). A detail from another GDNF-deprived neuron with double-membranedautophagosomes (ap) and single-membraned autolysosomes (al) containingswirled packages of undigested membranes. Scale bar: 1 μm.

FIGS. 8A, 8B, 8C, and 8D. Mitochondria of NGF-deprived, but notGDNF-deprived, sympathetic neurons are structurally changed. (8A)Typical view of a GDNF-deprived neuron with numerous dark autophagicprofiles and several nonclustered elongated mitochondria with normalcristae. (8B) A NGF-deprived neuron with several dark autolysosomes andlarge number of round clustered mitochondria with changed cristae. N:nucleus. (8C) Higher magnification of the mitochondrial cluster withvesicular cristae and one membrane in a NGF-deprived neuron. (8D)Mitochondria whose cristae and inner membrane are altered to differentextent in a NGF-deprived neuron. Scale bar: 1 μm. (8E) Distribution ofmitochondrial profiles from the sections of GDNF-deprived (n=201) andNGF-deprived (n=317) neurons according to their cross-sectional areas.Size categories are shown as percent of all mitochondrial profiles.

FIG. 9. GDNF deprivation does not induce chromatin condensation andnuclear fragmentation in the sympathetic neurons. Six DIV neurons weredeprived of or maintained with GDNF or NGF. The cultures were fixeddaily and stained with Hoechst 33258. The neurons with fragmented nucleiand condensed chromatin were counted and expressed as percent of allneurons. The mean±standard error of the mean of four independentexperiments is shown for each data point.

DETAILED DESCRIPTION OF THE INVENTION

Growth factors referred in this invention comprise growth factors whichposses activities for neuronal or nonneuronal cells. Growth factorsuseful in this invention comprise GDNF family members, different FGFs,IGFs, TGF-betas, EGFs, CNTF, LIF, NGF, BDNF, NT-4, NT-4 and AL-1.

GDNF family members comprise GDNF (glial cell line-derived neurotrophicfactor), NRTN (neurturin), ARTN (artemin) and PSPN (persephin).

As used herein, the term “neurons” or “neuronal cells” are intended toinclude the neurons of the central nervous system and peripheral nervoussystem. Preferred neurons are mammalian neurons, more preferably humanneurons.

As used herein, the term “nonneuronal” are intended to include cells asastrocytes, oligodendrocytes, kidney cells, chromaffin cells, T-cells,i.e. cells not encompassed in term “neuron”.

We describe here that removal of GDNF from the cultured sympatheticneurons triggers a novel nonmitochondrial caspase-dependent deathpathway. We have discovered that in GDNF-deprived apoptotic neurons, atleast caspase-2 and caspase-7 are activated. Thus, the present inventionprovides a method, which can be used in screening cell death modulatorsof neuronal or nonneuronal cells in a GDNF family growthfactor-dependent cell culture system, wherein candidate modulators areintroduced into the system and effective modulators are identified bydetermining the activity of caspase-2 and/or caspase-7 after GDNFdeprivation. Active modulators discovered by the method may be used indrug development.

Cell Culture Methods for Identifying Caspase-2 and/or Caspase-7Modulators

In one embodiment, the invention contemplates in vitro methods forculturing neuronal or nonneuronal cells under conditions where thecaspase-2 and/or caspase-7 modulating agent are used to prevent celldeath and can include methods for detecting the presence and amount ofmodulation of caspase-2 and/or caspase-7 activity.

Appropriate cells are prepared for identification of caspase-2 and/orcaspase-7 modulating agents in a growth factor deprivation assay. Forexample, a preparation of superior cervical ganglion neurons isdisclosed in the Experimental Section. Another appropriate cells aredorsal root ganglion cells, nodose ganglion neurons, spinal motoneurons,midbrain dopaminergic neurons, central noradrenergic neurons and entericneurons. The nonneuronal cells for the identification of caspase-2and/or caspase-7 modulating agents are chromaffin cells of the adrenalmedulla, cells of embryonic kidney, differentiating spermatogonia orT-cells of the thyroid gland.

Before beginning the assay, the cells may be resuspended, added tosubstrate-coated dishes, and placed under predetermined assay conditionsfor a preselected period of time. After the attachment and growthperiod, the dishes may be rinsed to remove unbound cells, fixed, andviewed e.g., by phase contrast microscopy.

Preferably, a plurality of cells are analysed for each substrate. Cellsare then “judged” based on predetermined criteria. For example, cellsmay be considered apoptotic if they show typical signs of apoptoticdeath. The percent of cells that are dying is preferably determined. Aparticularly preferred cell death assay method is disclosed in theExperimental Section.

In one embodiment, the invention provides a method of screening celldeath modulators of neuronal or nonneuronal cells in a GDNF familygrowth factor dependent cell culture system comprising the steps of (1)removing the GDNF family growth factor from the culture system; (2)introducing a candidate modulator agent into the culture system; and (3)determining the activity of caspase-2 and/or caspase-7. Steps 1 and 2can be performed in any order. Preferably, step 2 is performed beforestep 1, more preferably, step 1 and 2 are performed simultaneously. Step3 can be performed several times during the screening procedure.

The invention also discloses compositions comprising agents exhibiting acaspase-2 and/or caspase-7 modulating activity in substantially pureform.

In Vivo Methods for Prevention of Cell Death

The various caspase-2 and/or caspase-7 activity modulating agents arealso useful in a variety of therapeutic applications as describedherein.

The present therapeutic methods are useful in treating growth factordeprived neurons or nonneuronal cells associated with physical orsurgical trauma, infarction, toxin exposure, degenerative disease,malignant disease that affects peripheral or central neurons, or insurgical or transplantation methods in which neuronal cells from brain,spinal cord or dorsal root ganglia are exposed to reduced levels ofgrowth factors and require cell death preventive therapy. Such diseasesfurther include but are not limited to CNS lesions, gliosis, Parkinson'sdisease, Alzheimer's disease, neuronal degeneration, enteric diseases,kidney disease, immunogical diseases, diseases of chromaffin cells andthe like.

In treating growth factor deprivation, contacting a therapeuticcomposition of this invention with the deprived cells soon after injuryis particularly important for accelerating the rate and extent ofrecovery.

Thus the invention contemplates a method of preventing cell death ingrowth factor deprivation in a subject, or in selected tissues thereof,comprising administering to the subject or the tissue a physiologicallytolerable composition containing a therapeutically effective amount ofan agent of the present invention.

In preferred methods, the subject is a human patient.

In one embodiment, a cell undergoing growth factor deprivation may berepaired by administering a caspase-2 and/or caspase-7 modulating agent.

In a related embodiment, a caspase-2 and/or caspase-7 modulating agentis administered orally in a pharmaceutically accepted formula. Thetarget cell may reside in culture or in situ, i.e. within the naturalhost. For in situ applications, the compositions are added to a retainedphysiological fluid such as blood or synovial fluid. For CNSadministration, a variety of techniques are available for promotingtransfer of the therapeutic agent across the blood brain barrierincluding disruption by surgery or injection, drugs which transientlyopen adhesion contact between CNS vasculature endothelial cells, andcompounds which facilitate translocation through such cells. A caspase-2and/or caspase-7 modulating agent may also be amenable to directinjection or infusion, topical, intratracheal/nasal administration e.g.through aerosol, intraocularly, or within/on implants e.g. fibers e.g.collagen, osmotic pumps, grafts comprising appropriately transformedcells, etc. Generally, the amount administered will be empiricallydetermined, typically in the range of about 10 to 1000 micrograms/kg ofthe recipient and the concentration will generally be in the range ofabout 50 to 500 micrograms/ml in the dose administered. Other additivesmay be included, such as stabilizers, bactericides, etc. will be presentin conventional amounts.

Therapeutic compositions of the present invention may include aphysiologically tolerable carrier together with at least one caspase-2and/or caspase-7 modulating agent of this invention as described herein,dispersed therein as an active ingredient. In a preferred embodiment,the therapeutic composition is not immunogenic when administered to ahuman patient for therapeutic purposes.

For the sake of simplicity, the active agent of the therapeuticcompositions described herein shall be referred to as a “caspase-2and/or caspase-7 modulating agent”. It should be appreciated that thisterm is intended to encompass a variety of agents including such smallmolecules which include, but are not limited to, peptides,peptidomimetics (e.g., peptoids), amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e., including heteroorganicand organometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds.

Any of a variety of mammalian cells or neuronal cells can be treated bythe present method of treatment in vivo, including neuronal cells frombrain, CNS, peripheral nerves and the like. In a preferred embodimentthese cells are superior cervical ganglion cells. In another preferredembodiment these cells are dorsal root ganglion cells, nodose ganglionneurons, spinal motoneurons, midbrain dopaminergic neurons, centralnoradrenergic neurons and enteric neurons. In still another preferredembodiment these cells comprise chromaffin cells of the adrenal medulla,cells of embryonic kidney, differentiating spermatogonia or T-cells ofthe thyroid gland.

In addition, the cells can be from any of a variety of mammalianspecies, including human, mouse, chicken, and any other mammalianspecies, including the agricultural stock and non-domesticated mammals.

Therapy

The present invention also encompasses agents which modulate caspase-2and/or caspase-7 expression or activity. An agent may, for example, be asmall molecule. For example, such small molecules include, but are notlimited to, peptides, peptidomimetics (e.g., peptoids), amino acids,amino acid analogs, polynucleotides, polynucleotide analogs,nucleotides, nucleotide analogs, organic or inorganic compounds (i.e.,including heteroorganic and organometallic compounds) having a molecularweight less than about 10,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 5,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 1,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 500 grams per mole, and salts, esters,and other pharmaceutically acceptable forms of such compounds. It isunderstood that appropriate doses of small molecule agents depends upona number of factors within the ken of the ordinarily skilled physician,veterinarian, or researcher. The dose(s) of the small molecule willvary, for example, depending upon the identity, size, and condition ofthe subject or sample being treated, further depending upon the route bywhich the composition is to be administered, if applicable, and theeffect which the practitioner desires the small molecule to have uponthe nucleic acid or polypeptide of the invention. Exemplary dosesinclude milligram or microgram amounts of the small molecule perkilogram of subject or sample weight (e.g., about 1 microgram perkilogram to about 500 milligrams per kilogram, about 100 micrograms perkilogram to about 5 milligrams per kilogram, or about 1 microgram perkilogram to about 50 micrograms per kilogram. It is furthermoreunderstood that appropriate doses of a small molecule depend upon thepotency of the small molecule with respect to the expression or activityto be modulated. Such appropriate doses may be determined using theassays described herein. When one or more of these small molecules is tobe administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

Screening Assays

The subject methods include screens for agents which modulate caspase-2and/or caspase-7 interactions and methods for modulating theseinteractions. Caspase-2 and/or caspase-7 activation is found to regulatecell death, more particularly growth factor deprivation dependent celldeath. Accordingly, the invention provides methods for modulatingtargeted cell function comprising the step of modulating caspase-2and/or caspase-7 activation by contacting the cell with a modulatoragent of a caspase-2 and/or caspase-7 activation.

In another aspect, the invention provides methods of screening foragents which modulate caspase-2 and caspase-7 interactions. Thesemethods generally involve forming a mixture of a caspase-2 andcaspase-7-expressing cell and a candidate agent, and determining theeffect of the agent on the amount of caspase-2 and/or caspase-7expressed by the cell. The methods are amenable to automated,cost-effective high throughput screening of chemical libraries for leadcompounds. Identified reagents find use in the pharmaceutical industriesfor animal and human trials; for example, the reagents may bederivatized and rescreened in vitro and in vivo assays to optimizeactivity and minimize toxicity for pharmaceutical development. Morespecifically, neuronal cell based growth factor deprivation assay isdescribed in detail in the experimental section below.

The invention further provides methods (also referred to herein as“screening assays”) for identifying modulators, i.e., candidate or testcompounds or agents (e.g., peptides, peptidomimetics, peptoids, smallmolecules or other drugs) which bind to caspase-2 and/or caspase-7proteins, have a stimulatory or inhibitory effect on, for example,caspase-2 and/or caspase-7 expression or caspase-2 and/or caspase-7activity, or have a stimulatory or inhibitory effect on, for example,the expression or activity of an caspase-2 and/or caspase-7 substrate.Compounds thus identified can be used to modulate the activity ofcaspase-2 and/or caspase-7 in a therapeutic protocol, to elaborate thebiological function of the caspase-2 and/or caspase-7, or to identifycompounds that disrupt normal caspase-2 and/or caspase-7 interactions.The preferred caspase-2 and caspase-7 used in this embodiment are thehuman caspase-2 and caspase-7 of the present invention.

In one embodiment, the invention provides assays for screening candidateor test compounds which are substrates of an caspase-2 and/or caspase-7protein or polypeptide or biologically active portion thereof. Inanother embodiment the invention provides assays for screening candidateor test compounds which bind to or modulate the activity of an caspase-2and/or caspase-7 protein or polypeptide or biologically active portionthereof.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; peptoid libraries [libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive] (see, e.g., Zuckermann, R. N. etal. J. Med. Chem. 1994, 37: 2678-85); spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the ‘one-bead one-compound’ library method; andsynthetic library methods using affinity chromatography selection. Thebiological library and peptoid library approaches are limited to peptidelibraries, while the other four approaches are applicable to peptide,non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12:145).

In one embodiment, an assay is a cell-based assay in which a cell whichundergoes growth factor deprivation is contacted with a test compoundand the ability of the test compound to modulate caspase-2 and/orcaspase-7 activity is determined. Determining the ability of the testcompound to modulate caspase-2 and/or caspase-7 activity can beaccomplished by monitoring, for example, cell death, cell growth, cellattachment, neurite outgrowth, and cell chemotaxis. The cell, forexample, can be of mammalian origin, e.g., a neuronal cell. In preferredembodiment, caspase-2 and/or caspase-7 is expressed in neuronal cellsand the ability of the test compound to modulate caspase-2 and/orcaspase-7 activity is accomplished by monitoring cell death oralternatively, by monitoring caspase-2 and caspase-7 activation withWestern blot, immunohistochemical staining using anti caspase-2 orcaspase-7 antibodies, or fluorometric assays described in experimentalsection.

Determining the ability of the caspase-2 and/or caspase-7 or abiologically active fragment thereof, to bind to or interact with anagent can be accomplished by one of the methods described above fordetermining direct binding. In a preferred embodiment, determining theability of the caspase-2 and/or caspase-7 protein to bind to or interactwith an agent can be accomplished by determining the activity of thecaspase-2 and/or caspase-7. For example, the activity of the caspase-2and/or caspase-7 can be determined by detecting catalytic/enzymaticactivity of the caspase-2 and/or caspase-7 upon an appropriate substrate(for example a fluorometric assay using caspase-2 or caspase-7 specificsubstrates), detecting the induction of a reporter gene (recombinantcaspase-2 and/or caspase-7 gene products labelled with detectablemarker), or detecting a target-regulated cellular response (i.e., cellattachment, adhesion, growth, death or migration).

In yet another embodiment, an assay of the present invention is acell-free assay in which an caspase-2 and/or caspase-7 protein orbiologically active portion thereof is contacted with a test compoundand the ability of the test compound to bind to the caspase-2 and/orcaspase-7 protein or biologically active portion thereof is determined.Preferred biologically active portions of the caspase-2 and/or caspase-7proteins to be used in assays of the present invention include fragmentswhich posses sites of their enzymatic activity.

The caspase-2 and/or caspase-7 proteins of the invention can, in vivo,interact with one or more cellular macromolecules, such as proteins. Forthe purposes of this discussion, such cellular macromolecules arereferred to herein as “binding partners.” Compounds that disrupt suchinteractions can be useful in regulating the activity of the caspase-2and/or caspase-7. Such compounds can include, but are not limited tomolecules such as peptides and small molecules. Towards this purpose, inan alternative embodiment, the invention provides methods fordetermining the ability of the test compound to modulate the activity ofa caspase-2 and/or caspase-7 protein through modulation of the activityof a upstream effector molecule of an caspase-2 and/or caspase-7. Forexample, the activity of the effector molecule on an caspase-2 and/orcaspase-7 can be determined, or the binding of the effector to caspase-2and/or caspase-7 can be determined as previously described.

Assays for the Detection of the Ability of a Test Compound to ModulateExpression of Caspase-2 and/or Caspase-7

In another embodiment, modulators of caspase-2 and/or caspase-7expression are identified in a method wherein a cell is contacted with acandidate compound/agent and the expression of caspase-2 and/orcaspase-7 mRNA or protein in the cell is determined. The level ofexpression of caspase-2 and/or caspase-7 mRNA or protein in the presenceof the candidate compound is compared to the level of expression ofcaspase-2 and/or caspase-7 mRNA or protein in the absence of thecandidate compound. The candidate compound can then be identified as amodulator of caspase-2 and/or caspase-7 expression based on thiscomparison. For example, when expression of caspase-2 and/or caspase-7mRNA or protein is greater (statistically significantly greater) in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of caspase-2 and/or caspase-7mRNA or protein expression. Alternatively, when expression of caspase-2and/or caspase-7 mRNA or protein is less (statistically significantlyless) in the presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of caspase-2 and/orcaspase-7 mRNA or protein expression. The level of caspase-2 and/orcaspase-7 mRNA or protein expression in the cells can be determined bymethods described herein for detecting caspase-2 and/or caspase-7 mRNAor protein.

Combination Assays

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell free assay, and the abilityof the agent to modulate the activity of an caspase-2 and/or caspase-7protein can be confirmed in vivo, e.g., in an animal such as an animalmodel for CNS disorders, or for cellular transformation and/or neuronaldeath.

This invention farther pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., an caspase-2 and/or caspase-7 modulating agent,an antisense caspase-2 and/or caspase-7 nucleic acid molecule, ancaspase-2 and/or caspase-7-specific antibody, or an caspase-2 and/orcaspase-7-binding partner) can be used in an animal model to determinethe efficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal model to determine the mechanism of action of such an agent.Furthermore, this invention pertains to uses of novel agents identifiedby the above-described screening assays for treatments as describedherein.

The choice of assay format will be based primarily on the nature andtype of sensitivity/resistance protein being assayed. A skilled artisancan readily adapt protein activity assays for use in the presentinvention with the genes identified herein.

For example, activation of caspases (proteolytic cleavage of theinactive pro-caspase into smaller subunits) can be detected by Westernblotting. A 48 kD precursor of caspase-2 is cleaved into p16, p18 andp12, and a 33 kD precursor of caspase-7 is cleaved into P12 and p19. Theantibodies are commercially available (e.g. Santa Cruz Biotechnology).Caspase activation can also be detected by fluorometrically byFRET-technology (see below).

In a preferred embodiment of the invention, the activation of theGDNF-deprived cell death pathway in the cells can be checked by theactivation of caspase-2 and caspase-7 in the conditions where caspase-3(but also caspase-9 and caspase-8), as well as Bax, —are not activated.Caspase-2 is often activated also in the “classical” apoptosis butalways together with caspase-3 (and either caspase-9 or caspase-8). Inthe death pathway activated by GDNF deprivation, caspase-2 is activatedwithout the activation of caspase-3, -9, or -8. Bax protein, that isessential in “classical” apoptosis, is also not activated in the deathpathway triggered by GDNF deprivation.

The activation of specific caspases can be checked by fluorescenceresonance electron transfer (FRET)-technique using caspase-specificprobes (Takemoto K, Nagai T, Miyawaki A, Miura M. Spatio-temporalactivation of caspase revealed by indicator that is insensitive toenvironmental effects. J. Cell Biol. 2003; 160(2): 23543). In theseprobes, cyan fluorescent protein is linked to yellow fluorescent proteinby a peptide containing a cleavage site for the particular caspase. Whenactivated, the caspase cleaves the probe that changes the fluorescenceemission from cyan to yellow. The plasmids containing FRET probes can betransfected into the cells by different cell type-specific means.Fluorescence of the transfected cells can be monitored in dying cells byinverted microscope equipped with the fluorescence filters for cyan andyellow fluorescent proteins.

Thus, the present invention is also related to fluorescence resonanceenergy transfer (FRET) which refers to distance-dependent interactionbetween the electronically excited states of two dye molecules in whichexcitation energy is transferred from a donor molecule to an acceptormolecule without emission of a photon. Primary conditions for FRET are:(i) donor (source) and acceptor (target) molecules must be in closeproximity (typically 10-100 Å), (ii) the absorption spectrum of theacceptor must overlap the fluorescence emission spectrum of the donor.Probes labeled with such energy transfer coupled dyes are described,e.g., in U.S. Pat. No. 6,028,190. Any pair or pool of dyes capable ofFRET can be used in the present invention.

Furthermore, the activation of Bax in the dying cells can be checked bycell-permeable Bax-inhibiting peptides to Ku70, a protein that normallybinds to Bax and keeps it inactive in healthy cells (Sawada M, Hayes P,Matsuyama S. Cytoprotective membrane-permeable peptides designed fromthe Bax-binding domain of Ku70. Nat Cell Biol. 2003 April; 5(4): 352-7).These peptides block the apoptotic death where Bax is involved, but donot block the death induced by GDNF deprivation.

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, to provide additionaldetails with respect to its practice, are incorporated herein byreference.

The invention will be described in more detail in the experimentalsection. However, it will be appreciated that the methods of the presentinvention can be incorporated in the form of a variety of embodiments,only a few of which are disclosed herein. It will be apparent for thespecialist in the field that other embodiments exist and do not departfrom the spirit of the invention. Thus, the described embodiments areillustrative and should not be construed as restrictive.

EXPERIMENTAL SECTION

Materials and Methods

Culture of Sympathetic Neurons and the Survival Assays

Culture of the superior cervical ganglion (SCG) neurons was performed aspublished (Hamner et al., 2001; Sun et al., 2001; Lindahl et al., 2001).Briefly, the neurons of postnatal day-1-2 Han/Wi strain rats were grownsix days in vitro on polyornithine -laminin-coated dishes or glasscoverslips with 100 ng/ml of human GDNF (PeproTech) or 30 ng/ml of 2.5 Smouse NGF (Promega). 4-5 times more neurons were initially plated forGDNF-experiments compared to NGF-controls, so that by the day ofneurotrophic factor deprivation, the number of neurons in both groupswas similar. To reduce the number of nonneuronal cells, 1 μM cytosinearabinoside (Sigma-Aldrich) was always included next day after plating,but was not added to factor-deprived neurons. To deprive GDNF, thecultures were washed gently three times with GDNF-free culture medium.To remove NGF, the cultures were washed once with NGF-free medium andfunction-blocking anti-NGF antibodies (Roche) were added. The compoundsof interest were added and initial neurons were counted immediatelyafter neurotrophic factor deprivation. Living neurons were counted dailyby a “blind” experimenter not aware of the identity of experimentalgroups. The following compounds were assayed: broad-range caspaseinhibitor boc-aspartyl(OMe)-fluoromethylketone. (BAF) (Enzyme SystemsProducts) at 50 μM; pCPT-cAMP (Sigma-Aldrich) at 500 nM, V5 and I5peptides at 200 μM. When the compounds were dissolved in DMSO, the sameamount of DMSO was always added to the control cultures.

Microinjections

The neurons were pressure-microinjected with expression plasmids (50ng/μl) encoding the proteins of interest together with enhancedGFP-encoding plasmid (10 ng/μl) as an indicator of successful injection.The relevant empty vector (pcDNA3.1 or pCR3.1) without the insert, aswell as uninjected controls were always included. When neurotrophicfactor-deprived neurons were analysed, the factor-maintained uninjectedneurons were always included to show that the neurons do not die due topoor culture conditions. Neurons tolerating the injection procedure werecounted 4-6 h later according to the map drawn with the help of squaresscratched to the bottom of the culture dish, and considered as initialneurons. Next morning, the few living injected neurons that did not showGFP fluorescence, were subtracted from the initial neurons. An average,25-80 neurons were successfully injected per experimental point. Allexperiments were repeated at least three times on the independentcultures. The results were expressed as mean±the standard error of themean and were tested for the significance by either one-way ANOVA andpost hoc Tukey's honestly significant difference test, or two tailedStudent's t test with two sample unequal variance. The null hypothesiswas rejected at P<0.05.

The following expression plasmids were injected: full-length human Baxand full-length human Bcl-x_(L) expression plasmids, dominant negativeFADD plasmid, Cys287Ala mutant of caspase 9, Cys320Ser mutant of mousecaspase-2, and plasmid for Ku70. Also, the expression plasmids for humanXIAP (Yu et al., 2003) and dominant negative mutants of caspase-3,caspase-6, caspase-7 and caspase-8 (active center cysteine mutated toalanine in all cases) (Forcet et al., 2001) were used.

Immunocytochemistry

The neurons were grown on round glass coverslips, fixed with fresh 4%paraformaldechyde in phosphate-buffered saline, permeabilized with 1%Triton X-100 and blocked with 5% of donkey serum (Jackson ImmunoResearchLaboratories) in PBS. Antibodies to the following antigens were used:cytochrome c (65971A; BD PharMingen), phosphorylated serine 63 of c-Jun(9261; Cell Signaling Technology), phosphorylated serine 73 of c-Jun(9169; Cell Signaling Technology). Cy3-conjugated secondary antibodies(Jackson ImmunoResearch) were used for visualization and the specimenswere mounted in Vectashield (Vector Laboratories). Images were capturedat room temperature with DM-IRB inverted microscope (Leica) using PLFLUOTAR objective (63 x/0.70) or, for FIG. 2 A, HC PL FLUOTAR (10x/0.30), a 3CCD color video camera (model DXC-950P) (Sony) under thecontrol of Image-Pro Plus software version 3.0 (Media Cybernetics).Individual images were processed and assembled with Photoshop 7.0 (AdobeSystems). In some experiments, the fixed cultures were treated with 1μg/ml of Hoechst 33258 (Molecular Probes) for 15 minutes, then mountedand observed for the nuclear morphology.

Electron Microscopy

The neurons were grown in the four-well plates (Nunc) and deprived ofneurotrophic factors. The medium of the control neurons was also changedand the factors were added back. The cultures were fixed 48 h afterneurotrophic factor deprivation with 2% of glutaraldehyde. To avoiddetachment of loosely attached dying neurons, fixative with two-foldstrength was carefully added to the culture medium. The cultures werethen processed for transmission electron microscopy as described (Yu etal., 2003). From each sample, we chose one section cut from the middlethird of the depth of the neuron, and all neurons within that sectionwere analysed by FEI Tecnai F12 transmission electron microscope(Philips Electron Optics, Holland), operated at 80 kV. In twoindependent experiments, altogether 39 GDNF-deprived, 63 NGF-deprived,25 GDNF-maintained and 39 NGF-maintained neurons were analysed. Verysimilar results were obtained from two independent experiments. Forestimation of the size of mitochondria from GDNF- or NGF-deprivedneurons, mitochondria at a final magnification of x 33,000 were manuallytraced onto transparencies that were scanned. Cross-sectional area ofthe mitochondria was measured using Image-Pro Plus version 3.0.

Western Blotting

The neurons were grown with GDNF or NGF for 6 days, then either deprivedfrom these factors or not deprived for 14-18 h or 48 h in the presenceof caspase inhibitor BAF. The cells were collected in the Laemmli samplebuffer, lysed for 1 h on ice and analysed for Westen blotting usingstandard techniques. The filters were sequentially probed withantibodies to c-Jun (sc-45; Santa Cruz Biotechnology, Inc.),phosphorylated serine 63 of c-Jun (Cell Signaling Technology) andphosphorylated serine 73 of c-Jun (Cell Signaling Technology).

Results

One Third of Neonatal Rat Superior Cervical Ganglion Neurons areGDNF-Responsive

When sympathetic neurons from one- or two-day-old rat superior cervicalganglion (SCG) were cultured with GDNF, the number of neurons graduallyand slowly decreased. By sixth day in vitro (DIV), about 34% of theinitially plated neurons had survived and this number did not decreasefurther (FIG. 1 A). In similar conditions, about 98% of theNGF-maintained neurons had survived (FIG. 1 A). Dose-dependence studyrevealed that 100 ng/ml was a saturating concentration of GDNF for SCGneurons (FIG. 1 B). Removal of GDNF from the six DIV cultures leads todeath of the neurons that was kinetically similar to the death ofNGF-deprived neurons (FIG. 1 C). In all studies described below, theneurons from newborn rat SCG were maintained in sister dishes with GDNF(100 ng/ml) or NGF (30 ng/ml) for six DIV, then deprived of thesefactors for 48 or 72 hours, and analysed. NGF- and GDNF-responsiveneurons were always analysed in parallel and received identicaltreatment. The GDNF-responsive neurons appeared morphologicallyindistinguishable from NGF-responsive neurons. However, the dataobtained from GDNF-responsive neurons were generally more variable, andmore cultures failed, when compared to NGF-responsive neurons. Also,small number of neurons in the GDNF-deprived dishes seemed to dienon-specifically due to the washing procedure. Thus, GDNF-responsiveneurons seem to be more sensitive to small variations in the cultureconditions, than NGF-responsive neurons.

Mitochondrial Death Pathway is Not Activated in GDNF-DeprivedSympathetic Neurons

To study the localization of cytochrome c, we removed GDNF or NGF fromthe respective neurons for 48 h in the presence of broad-range caspaseinhibitor BAF, and stained the neurons with anti-cytochrome cantibodies. Only a small number of GDNF-deprived neurons showed faintdiffuse cytochrome c staining characteristic of its cytosoliclocalization (FIG. 2). In contrast, removal of NGF dramatically reducedthe number of neurons, with punctate mitochondrial cytochrome clocalization (FIG. 2), as shown by others (Deshmukh and Johnson, 1998;Neame et al., 1998; Martinou et al., 1999). Similar results wereobtained at 72 h after neurotrophic factor deprivation (not shown).

The role of Bax in the GDNF-deprived neurons was studied byoverexpressing Ku70, a protein that was recently shown to bind theN-terminus of Bax, thereby inhibiting its translocation to themitochondria (Sawada et al., 2003a; Sawada et al., 2003b). Ku70 had noeffect on the death of GDNF-deprived neurons, although it significantlyblocked the death of NGF-deprived neurons at 72 hours (FIG. 3 A).Similar effect was achieved with the Ku70-derived cell permeable peptideV5 shown to block the activity of Bax (Sawada et al., 2003a), whereasthe control peptide I5 had no effect (FIG. 3 B).

Involvement of caspase-9 and caspase-3 in the death of neurotrophicfactor-deprived sympathetic neurons was then investigated. However,direct demonstration of activation of the caspases by Western blottingappeared impossible due to scarcity of the material. Therefore weoverexpressed dominant negative mutants of these caspases in GDNF- orNGF-deprived neurons. Inhibition of either caspase-9 or caspase-3 failedto inhibit the death of GDNF-deprived neurons, although they efficientlyblocked death of NGF-deprived neurons by 72 h after microinjection andNGF deprivation (FIG. 4).

The absence of cytochrome c release together with a failure to observethe role of Bax, caspase-9 and caspase-3 suggest that GDNF-deprivedneurons die via a nonmitochondrial pathway. To confirm this, weoverexpressed the anti-apoptotic Bcl-2 family member Bcl-x_(L), shown toblock the mitochondrial death pathway, in the GDNF- and NGF-deprivedneurons. Overexpressed Bcl-x_(L) did not rescue GDNF-deprived neurons(FIG. 5 A), further confirming that the mitochondrial death pathway isnot activated. Bcl-x_(L), however, efficiently blocked the death ofNGF-deprived neurons (FIG. 5 A), as also shown by others(Gonzalez-Garcia et al., 1995). GDNF-maintained neurons were killed byoverexpressed pro-apoptotic protein Bax (FIG. 5 A), and Bax acceleratedthe death of GDNF-deprived neurons (not shown).

Caspase-2 and Caspase-7 are Activated in GDNF-Deprived SympatheticNeurons

The broad-range caspase inhibitor BAF almost completely blocked death ofboth GDNF-deprived (FIG. 5 B) and NGF-deprived (FIG. 5 B) (Deshmukh etal., 1996; Martinou et al., 1999; Deshmukh et al., 2000) neurons showingthat some caspases are absolutely required for the death ofGDNF-deprived neurons. To identify the relevant caspases, weoverexpressed by microinjection the dominant negative mutants ofcaspase-2, -3, -6, -7 and -8 in the GDNF-deprived, but also NGF-deprivedneurons. Blocking of caspase-2 and, to lesser extent, caspase-6 andcaspase-7 significantly inhibited death of NGF-deprived neurons, whereasdominant negative caspase-8 had no effect (FIG. 4). A dominant negativemutant of caspase-2, and to lesser extent, caspase-7, also inhibiteddeath of GDNF-deprived neurons, whereas blocking of caspase-6 andcaspase-8 had no effect (FIG. 4). Thus, GDNF deprivation-induced deathrequires caspase-2 and caspase-7 in sympathetic neurons.

Caspase-2 was recently shown to be activated upstream of themitochondria, and this event was required for the permeabilization ofthe mitochondria (Lassus et al., 2002). The presence of BAF in ourculture medium may thus, via inhibition of caspase-2, block cytochrome crelease in GDNF-deprived neurons and force them to chose anotherpathway. We therefore deprived the neurons of neurotrophic factorswithout BAF and applied cytochrome c immunocytochemistry. Although manyneurons have disappeared, cytochrome c immunostaining was still mostlypunctate in the remaining GDNF-deprived neurons (and in about half ofthe remained NGF-deprived neurons) (FIG. 2 C).

We also overexpressed the X chromosome-linked Inhibitor of ApoptosisProtein (XIAP), a natural inhibitor of caspases, in GDNF- andNGF-deprived neurons. XIAP did not affect the death of GDNF-deprivedneurons (FIG. 5 C) although, as expected (Yu et al., 2003), it rescuedsignificant portion of NGF-deprived neurons (FIG. 5 C). Overexpressionof XIAP did not significantly affect the viability of NGF-maintained orGDNF-maintained neurons (not shown). Thus, the caspases that executedeath of GDNF-deprived neurons could not be blocked by overexpressedXIAP.

c-Jun is Required for the Death of, and is Differently Activated inGDNF-Deprived and NGF-Deprived Sympathetic Neurons

To study the activation of the transcription factor c-Jun, we deprivedGDNF- or NGF-responsive BAF-saved neurons from the respective factorsfor 48 h, stained the neurons with antibodies to phosphorylated serines63 or 73, and counted the neurons with strong nuclear immunoreactivity.Deprivation of GDNF significantly increased the number of neuronsimmunopositive for phosphorylated serine 73 (FIG. 6 A). However, thenumber of nuclei positive for phosphorylated serine 63 was unchanged inthe GDNF-deprived neurons (FIG. 6 A, B). In control cultures,deprivation of NGF dramatically induced phosphorylation of serine 63 ofc-Jun, as shown by others (Ham et al., 1995; Virdee et al., 1997; Eilerset al., 1998; Harris et al., 2002), but also of serine 73 (FIG. 6 A, B)(Besirli and Johnson, 2003). As expected, only faint staining wasobtained for the neurons maintained in the presence of GDNF or NGF (FIG.6 A). Similar results were obtained at 72 h after neurotrophic factordeprivation (not shown).

In summary, our data show that activation of c-Jun is necessary for thedeath of GDNF-deprived neurons, although it is activated differentlyfrom NGF-deprived neurons.

Death Receptor Pathway is not Activated in GDNF-Deprived SympatheticNeurons

The data presented above show that GDNF-deprived sympathetic neurons dievia a nonmitochondrial death pathway where c-Jun, as well as caspase-2and caspase-7 are involved. Another well-characterized pathway, thedeath receptor-mediated pathway can be efficiently blocked by dominantnegative mutant of FADD/MORT1, an adapter that links pro-caspase-8 tomost death receptors (Vincenz, 2001; Strasser and Newton, 1999).Overexpression of this mutant FADD in GDNF-deprived, but also inNGF-deprived sympathetic neurons did not change the death rate (FIG. 5D), neither did it affect the viability of neurons maintained with NGFor GDNF (not shown). Also, as shown above, overexpression of dominantnegative caspase-8 did not affect death of neurons deprived of eitherfactor (FIG. 4). Thus, the death receptor pathway is probably notactivated in the NGF- or GDNF-deprived sympathetic neurons. Inaccordance with that, activation of death receptors tumor necrosisfactor-α or Fas on the surface of NGF-maintained sympathetic neurons didnot induce their death Putcha et al., 2002).

Ultrastructure of GDNF-Deprived and NGF-Deprived Sympathetic Neurons

To investigate the ultrastructural changes caused by removal of GDNF orNGF in sympathetic neurons, we deprived the cultures of neurotrophicfactors for 48 h and analysed the neurons by transmission electronmicrsocopy. Neurons maintained with GDNF or NGF were analysed as well.As a general observation, the cytoplasm of GDNF-deprived neurons wasmuch more electron-dense than that of NGF-deprived neurons in the sisterculture or the neurons maintained with either neurotrophic factor.Removal of GDNF led to marked increase in the number of differentautophagic profiles, including double-membraned autophagosomes andsingle-membraned autolysosomes that often contained swirled packs ofundigested membranes (FIG. 7). Thus, on average, 9 autolysosomes perneuron but no autophagosomes were found in GDNF-maintained neurons(n=25), whereas, on average, 3 autophagosomes and 14 autolysosomes werefound per GDNF-deprived neuron (n=39). NGF-deprived neurons alsoexhibited an increased number of autolysosomes (average of 18 perNGF-deprived neuron; n=63 versus 9 per NGF-maintained neuron; n=39) butthe autophagosomes were only rarely found (0.3 per average NGF-deprivedneuron and none in NGF-maintained neurons). Normal endoplasmic reticulumand Golgi complex were still found in the GDNF- and NGF-deprived neuronswith enhanced autophagy.

The mitochondria of GDNF-deprived neurons (FIG. 8 A) were similar tothose of GDNF-maintained (and also NGF-maintained) neurons (not shown),having mostly the orthodox configuration including elongated shape andclear cristae, and were not clustered. Such normal mitochondria werefound in all GDNF-deprived neurons, including those with massiveautophagy (FIG. 8 A). However, the mitochondria in NGF-deprived neuronswere often round-shaped and clustered (FIG. 8 B). The cristae of suchmitochondria were markedly reorganized, often observed to be round,vesicular and rare in the number, and sometimes only one mitochondrialmembrane was discernible (FIG. 8 C). Some mitochondria in these clusterscontained different numbers of normal rod-like cristae along with themitochondria with changed vesicular cristae (FIG. 8 D). In many neuronswith such mitochondrial clusters, few elongated nonclusteredmitochondria with the orthodox configuration were found, whereas someneurons contained only normal mitochondria (not shown). Of allNGF-deprived neurons analysed in two experiments (n=63), 43% hadround-shaped, clustered mitochondria with changed cristae, 19% had themitochondria in orthodox configuration, and in 38%, both types ofmitochondria were found. Appearance of the round clustered mitochondriaseems not to be a feature of final death phase, as these were oftenfound in neurons with normal endoplasmic reticulum, Golgi complex, andnucleus without condensed chromatin. We did not observe swelling anddisruption of the mitochondria in NGF-deprived neurons.

The average cross-sectional area of the mitochondria was 0.0781±0.0039μm² (mean±S.E.M., n=201) in the GDNF-deprived neurons and 0.038±0.0013μm² (n=317) in the NGF-deprived neurons. These values, as determinedfrom thin sections, do not directly indicate the length or size of themitochondria. In each section there is a collection of profiles fromsmallest perpendicular round profiles to longer oval-shape or evenbranched profiles depending of the orientation of the mitochondria inrelation to sectioning angle. To illustrate the difference in the sizeof the mitochondria we plotted the distribution of profiles according totheir cross-sectional area (FIG. 8 E). About 50% of all profiles insections from the NGF-deprived neurons fit into category of 0.02-0.04μm² and there were few profiles larger than 0.12 μm², whereas broadrange of mitochondrial profiles up to 0.3 μm² and larger were found fromthe GDNF-deprived neurons.

We did not found nuclei with condensed chromatin in the GDNF-deprivedneurons, analysed by electron microscopy, although 24% of theNGF-deprived neurons had the nuclei with DNA condensed to differentextent (not shown). We also stained the neurons maintained with ordeprived of GDNF or NGF with Hoechst 33258 and counted the neuronshaving typical fragmented nuclei with condensed chromatin. As shown onFIG. 9, progressively increasing number of NGF-deprived neurons withapoptotic nuclei was observed during three-day period, whereas onlysmall fraction (5-7%) of the GDNF-deprived or -maintained neurons hadfragmented nuclei. This number did not increase with time and mostprobably shows nonspecific death. Virtually all NGF-maintained neuronshad normal nuclei (not shown). Thus, we did not observe nuclear changesin the GDNF-deprived neurons.

Discussion

We found that removal of GDNF from the cultured sympathetic neuronstriggers a novel nonmitochondrial MLK-, c-Jun- and caspase-dependentdeath pathway, although removal of NGF from the sympathetic neuronsactivates the mitochondrial pathway. This is to our knowledge, the firstdescription of a nonmitochondrial pathway activated by withdrawal of asurvival factor.

GDNF Deprivation-Induced Death Requires MLK-c-Jun Pathway, Caspases-2and -7, and Involves Increased Autophagy

The mitochondrial pathway is not activated in GDNF-deprived sympatheticneurons. Indeed, cytochrome c is not released from the mitochondria tocytosol, Bax, caspases-9 and -3 are not involved in death execution,overexpression of Bcl-x_(L) does not protect the neurons, and theultrastructure of the mitochondria in GDNF-deprived neurons is notchanged. We have currently found several proteins involved in the deathof GDNF-deprived neurons. MLK and c-Jun are activated and seem to besimilarly required for death of both NGF- and GDNF-deprived neurons.Surprisingly we did not detect increase in phospho-serine 63immunoreactivity of c-Jun in GDNF-deprived neurons, although bothserines 63 and 73 were phoshorylated in NGF-deprived neurons. Thekinases that phosphorylate c-Jun in GDNF-deprived neurons remain to bestudied.

Caspase-2 and caspase-7 are involved in the death of GDNF-deprivedsympathetic neurons. Very little is known about the mechanism ofcaspase-2 activation. However, it is tempting to speculate thatcaspase-2 functions as an initiator and caspase-7 an executioner inthese neurons. Recent reports that caspase-2 is activated upstream ofmitochondrial events in some apoptotic cell types (Guo et al., 2002;Lassus et al., 2002; Read et al., 2002) are in accordance with our data.Overexpressed XIAP did not rescue GDNF-deprived neurons, although XIAPcan inactivate caspase-7 in cell-free systems (Deveraux et al., 1997)suggesting that caspase-7 is not available for XIAP in theGDNF-responsive sympathetic neurons. It should also be stressed that,although the dominant-negative caspase isoforms used here should bespecific for given caspases, some non-specific effects cannot becompletely excluded.

Many cells in which the main mitochondrial death pathway is geneticallyor pharmacologically disabled can still die via an alternative,autophagic pathway that is often caspase-independent and with increasedautophagy leading to largely vacuolised cytoplasm (Sperandio et al.,2000; Yaginuma et al., 2001; Oppenheim et al., 2001; Zaidi et al., 2001;Marsden et al., 2002). Dying GDNF-deprived neurons, however, seem todiffer from those “classical” autophagic death patterns, as the caspasesare clearly involved. We indeed observed markedly increased autophagy inthe GDNF-deprived neurons, but no remarkable vacuolisation of thecytoplasm was found, at least before the short final death executionphase. Few neurons in the terminal state of death that were retained inour electron microscopic preparations showed typical features ofsecondary necrosis, similar for both GDNF- and NGF-deprived neurons(data not shown). We found increased autophagy also in the NGF-deprivedneurons, as described by others (Martinou et al., 1999; Xue et al.,1999; Kirkland et al., 2002). Thus, both NGF- and GDNF-deprived neuronsdie in a caspase-dependent manner with enhanced autophagy.

NGF-Deprived Sympathetic Neurons Die via Mitochondrial Pathway

We confirmed the published data that NGF-deprived neurons die via themitochondrial pathway, including cytosolic localization of cytochrome c(Deshmukh and Johnson, 1998; Neame et al., 1998; Martinou et al., 1999),involvement of Bax (Deckwerth et al., 1996; Putcha et al., 1999),caspase-9 and caspase-3 (Deshmukh et al., 2000; Deshmukh et al., 2002)and inhibition of death by Bcl-x_(L) (Gonzalez-Garcia et al., 1995). Inaddition, we showed for the first time that also caspase-6 and caspase-7are necessary for NGF deprivation-induced death, and confirmed the roleof caspase-2 (Troy et al., 2001).

Our overexpression studies (Yu et al., 2003); this study) are inagreement with the current concept that in NGF-responsive sympatheticneurons, critical caspases are blocked with Inhibitor of ApoptosisProteins, e.g. XIAP. Withdrawal of NGF releases caspases from that blockby proteasome-mediated degradation, but also by removal of XIAP from thecaspases by Smac/DIABLO that is released from the mitochondria togetherwith cytochrome c (Troy et al., 2001; Deshmukh et al., 2002; Yu et al.,2003). Most probably a large amount of overexpressed XIAP replaces thedegraded bulk and keeps the caspases inactivated in our experiment.

We found remarkable ultrastructural changes in the mitochondria of manyNGF-deprived neurons: they gradually become round, clustered and theircristae changed considerably. Appearance of round mitochondria as aresult of increased fission has been described previously in theNGF-deprived neurons (Martinou et al., 1999) and in other cells(Karbowski et al., 2002). Also, the clustering of mitochondria inNGF-deprived neurons has been described (Tolkovsky et al., 2002),although the mechanism remained obscure. However, our observation thatthe cristae in the small clustered mitochondria of NGF-deprived neuronswere often round, vesicular, and reduced whereas the inner membrane wassometimes found to be missing, have not been described in other studies(Martin et al., 1988; Martinou et al., 1999; Xue et al., 1999; Kirklandet al., 2002). We do not know whether this discrepancy results fromdifferences in the culture conditions, genetic background of theanimals, or from other conditions, but this ultrastructural pattern wasrepeatedly observed in our cultures. Mitochondria with orthodox andaltered ultrastructure were found in the same sample, sometimes even inthe same neuron, ruling out the possibility of a processing artifact.The mitochondrial cristae are dynamic structures that can considerablychange their shape (Frey et al., 2002). In the apoptotic cells, thesechanges are proposed to facilitate the release of cristae-associatedcytochrome c into the intermembrane space (Scorrano et al., 2002). It istempting to speculate that the mitochondria with altered cristae havealready released their cytochrome c. We also stress that our data do notsupport the release of apoptotic proteins from the mitochondria viatheir swelling and rupture.

In summary, we propose that GDNF-deprived sympathetic neurons die bycaspase-dependent nonmitochondrial death pathway that has not beendescribed previously. More studies are required to characterize themolecular and cellular components of this pathway. How an exposure ofSCG neurons to different neurotrophic factors dictates the deathprogram, is currently unknown. It was recently shown that an apoptoticfragment, generated from unligated Ret by caspase-3, can triggerapoptosis in some cell lines (Bordeaux et al., 2000). However,overexpression of Ret or apoptotic fragment of Ret in the sympatheticneurons did not induce their death in our model (unpublished data),suggesting that death-promoting activity of unligated Ret is notmanifested in the sympathetic neurons. Ret, similarly to Deleted inColorectal Cancer (Forcet et al., 2001), may be able to recruit andactivate caspases directly, so that mitochondrial pathway is notrequired. Alternatively, exposure of the neurons to GDNF for six daysmay differentiate the neurons so that the mitochondrial pathway isnonfunctional. Whether and how the nonmitochondrial death pathway isused in vivo is currently unknown, as virtually nothing is yet knownabout the biological role of GDNF for the SCG neurons.

Some of the abbreviations used herein: NGF, nerve growth factor; GDNF,glial cell line-derived neurotrophif factor; SCG, superior cervicalganglion; DIV, days in vitro; BAF, boc-aspartyl(OMe)-fluoromethylketone;XIAP, X chromosome-linked inhibitor of apoptosis protein; BDNF,brain-derived neurotrophic factor.

REFERENCES

-   1. Airaksinen, M. S. and M. Saarma. 2002. The GDNF family:    signalling, biological functions and therapeutic value. Nat. Rev.    Neurosci. 3:383-394.-   2. Besirli, C. G. and E. M. Johnson, Jr. 2003. JNK-independent    activation of c-Jun during neuronal apoptosis Induced by multiple    DNA-damaging agents. J. Biol. Chem. 278:22357-22366.-   3. Bordeaux, M. C., C. Forcet, L. Granger, V. Corset, C. Bidaud, M.    Billaud, D. E. Bredesen, P. Edery, and P. Mehlen. 2000. The RET    proto-oncogene induces apoptosis: a novel mechanism for Hirschsprung    disease. EMBO J. 19:4056-4063.-   4. Clarke, P. G. 1990. Developmental cell death: morphological    diversity and multiple mechanisms. Anat. Embryol. (Berl)    181:195-213.-   5. Deckwerth, T. L., J. L. Elliott, C. M. Knudson, E. M. Johnson,    Jr., W. D. Snider, and S. S. Korsmeyer. 1996. BAX is required for    neuronal death after trophic factor deprivation and during    development. Neuron 17:401-411.-   6. Deshmukh, M. and E. M. Johnson, Jr. 1998. Evidence of a novel    event during neuronal death: development of competence-to-die in    response to cytoplasmic cytochrome c. Neuron 21:695-705.-   7. Deshmukh, M., J. Vasilakos, T. L. Deckwerth, P. A. Lampe, B. D.    Shivers, and E. M. Johnson, Jr. 1996. Genetic and metabolic status    of NGF-deprived sympathetic neurons saved by an inhibitor of ICE    family proteases. J. Cell Biol. 135:1341-1354.-   8. Deshmukh, M., C. Du, X. Wang, and E. M. Johnson, Jr. 2002.    Exogenous Smac induces competence and permits caspase activation in    sympathetic neurons. J. Neurosci. 22:8018-8027.-   9. Deshmukh, M., K. Kuida, and E. M. Johnson. 2000. Caspase    inhibition extends the commitment to neuronal death beyond    cytochrome c release to the point of mitochondrial    depolarization. J. Cell Biol. 150:131-144.-   10. Deveraux, Q. L., R. Takahashi, G. S. Salvesen, and J. C.    Reed. 1997. X-linked IAP is a direct inhibitor of cell-death    proteases. Nature 388:300-304.-   11. Edwards, S. N. and A. M. Tolkovsky. 1994. Characterization of    apoptosis in cultured rat sympathetic neurons after nerve growth    factor withdrawal. J. Cell Biol. 124:537-546.-   12. Eilers, A., J. Whitfield, C. Babij, L. L. Rubin, and J.    Ham. 1998. Role of the Jun kinase pathway in the regulation of c-Jun    expression and apoptosis in sympathetic neurons. J. Neurosci.    18:1713-1724.-   13. Ellerby, L. M., A. S. Hackam, S. S. Propp, H. M. Ellerby, S.    Rabizadeh, N. R. Cashman, M. A. Trifiro, L. Pinsky, C. L.    Wellington, G. S. Salvesen, M. R. Hayden, and D. E. Bredesen. 1999.    Kennedy's disease: caspase cleavage of the androgen receptor is a    crucial event in cytotoxicity. J. Neurochem. 72:185-195.-   14. Estus, S., W. J. Zaks, R. S. Freeman, M. Gruda, R. Bravo,    and E. M. Johnson, Jr. 1994. Altered gene expression in neurons    during programmed cell death: identification of c-jun as necessary    for neuronal apoptosis. J. Cell Biol. 127:1717-1727.-   15. Forcet, C., X. Ye, L. Granger, V. Corset, H. Shin, D. E.    Bredesen, and P. Mehlen. 2001. The dependence receptor DCC (deleted    in colorectal cancer) defines an alternative mechanism for caspase    activation. PNAS 98:3416-3421.-   16. Frey, T. G., C. W. Renken, and G. A. Perkins. 2002. Insight into    mitochondrial structure and function from electron tomography.    Biochimica et Biophysica Acta (BBA)—Bioenergetics 1555:196-203.-   17. Gonzalez-Garcia, M., I. Garcia, L. Ding, S. O'Shea, L. H.    Boise, C. B. Thompson, and G. Nunez. 1995. bcl-x is expressed in    embryonic and postnatal neural tissues and functions to prevent    neuronal cell death. PNAS 92:4304-4308.-   18. Guo, Y., S. M. Srinivasula, A. Druilhe, T. Fernandes-Alnemri,    and E. S. Alnemri. 2002. Caspase-2 induces apoptosis by releasing    proapoptotic proteins from mitochondria. J. Biol. Chem.    277:13430-13437.-   19. Ham, J., C. Babij, J. Whiftield, C. M. Pfarr, D. Lallemand, M.    Yaniv, and L. L. Rubin. 1995. A c-Jun dominant negative mutant    protects sympathetic neurons against programmed cell death. Neuron    14:927-939.-   20. Hamner, S., U. Arumäe, Y. Li-Ying, Y. F. Sun, M. Saarma, and D.    Lindholm. 2001. Functional characterization of two splice variants    of rat bad and their interaction with Bcl-w in sympathetic neurons.    Mol. Cell Neurosci. 17:97-106.-   21. Harris, C. A., M. Deshmukh, B. Tsui-Pierchala, A. C. Maroney,    and E. M. Johnson, Jr. 2002. Inhibition of the c-Jun N-terminal    kinase signaling pathway by the mixed lineage kinase inhibitor    CEP-1347 (KT7515) preserves metabolism and growth of trophic    factor-deprived neurons. J. Neurosci. 22:103-113.-   22. Huang, E. J. and L. F. Reichardt. 2001. Neurotrophins: roles in    neuronal development and function. Annu. Rev. Neurosci. 24:677-736.-   23. Karbowski, M., Y. J. Lee, B. Gaume, S. Y. Jeong, S. Frank, A.    Nechushtan, A. Santel, M. Fuller, C. L. Smith, and R. J.    Youle. 2002. Spatial and temporal association of Bax with    mitochondrial fission sites, Drp1, and Mfn2 during apoptosis. J.    Cell Biol. 159:931-938.-   24. Kirkland, R. A., R. M. Adibhatla, J. F. Hatcher, and J. L.    Franklin. 2002. Loss of cardiolipin and mitochondria during    programmed neuronal death: evidence of a role for lipid peroxidation    and autophagy. Neuroscience 115:587-602.-   25. Kotzbauer, P. T., P. A. Lampe, R. O. Heuckeroth, J. P.    Golden, D. J. Creedon, E. M. Johnson, Jr., and J. Milbrandt. 1996.    Neurturin, a relative of glial-cell-line-derived neurotrophic    factor. Nature 384:467-470.-   26. Lassus, P., X. Opitz-Araya, and Y. Lazebnik. 2002. Requirement    for caspase-2 in stress-induced apoptosis before mitochondrial    permeabilization. Science 297:1352-1354.-   27. Leist, M. and M. Jäättelä. 2001. Four deaths and a funeral: from    caspases to alternative mechanisms. Nat. Rev. Mol. Cell Biol.    2:589-598.-   28. Lindahl, M., D. Poteryaev, L. Yu, U. Arumäe, T. Timmusk, I.    Bongarzone, A. Aiello, M. A. Pierotti, M. S. Airaksinen, and M.    Saarma. 2001. Human glial cell line-derived neurotrophic factor    receptor alpha 4 is the receptor for persephin and is predominantly    expressed in normal and malignant thyroid medullary cells. J. Biol.    Chem. 276:9344-93511.-   29. Llambi F., F. Causeret, E. Bloch-Gallego, and P. Mehlen. 2001.    Netrin-1 acts as a survival factor via its receptors UNC5H and DCC.    EMBO J. 20:2715.-   30. Maroney, A. C., J. P. Finn, D. Bozyczko-Coyne, T. M.    O'Kane, N. T. Neff, A. M. Tolkovsky, D. S. Park, C. Y. Yan, C. M.    Troy, and L. A. Greene. 1999. CEP-1347 (KT7515), an inhibitor of JNK    activation, rescues sympathetic neurons and neuronally    differentiated PC12 cells from death evoked by three distinct    insults. J. Neurochem. 73:1901-1912.-   31. Maroney, A. C., J. P. Finn, T. J. Connors, J. T. Durkin, T.    Angeles, G. Gessner, Z. Xu, S. L. Meyer, M. J. Savage, L. A.    Greene, R. W. Scott, and J. L. Vaught. 2001. CEP-1347 (KT7515), a    semisynthetic inhibitor of the mixed lineage kinase family. J. Biol.    Chem. 276:25302-25308.-   32. Marsden, V. S., L. O'Connor, L. A. O'Reilly, J. Silke, D.    Metcalf, P. G. Ekert, D. C. Huang, F. Cecconi, K. Kuida, K. J.    Tomaselli, S. Roy, D. W. Nicholson, D. L. Vaux, P. Bouillet, J. M.    Adams, and A. Strasser. 2002. Apoptosis initiated by Bcl-2-regulated    caspase activation independently of the cytochrome    c/Apaf-1/caspase-9 apoptosome. Nature 419:634-637.-   33. Martin, D. P., R. E. Schmidt, P. S. DiStefano, O. H.    Lowry, J. G. Carter, and E. M. Johnson, Jr. 1988. Inhibitors of    protein synthesis and RNA synthesis prevent neuronal death caused by    nerve growth factor deprivation. J. Cell Biol. 106:829-844.-   34. Martinou, I., S. Desagher, R. Eskes, B. Antonsson, E. Andre, S.    Fakan, and J. C. Martinou. 1999. The release of cytochrome c from    mitochondria during apoptosis of NGF-deprived sympathetic neurons is    a reversible event. J. Cell Biol. 144:883-889.-   35. Neame, S. J., L. L. Rubin, and K. L. Philpott. 1998. Blocking    cytochrome c activity within intact neurons inhibits apoptosis. J.    Cell Biol. 142:1583-1593.-   36. Oppenheim, R. W., R. A. Flavell, S. Vinsant, D. Prevette, C. Y.    Kuan, and P. Rakic. 2001. Programmed cell death of developing    mammalian neurons after genetic deletion of caspases. J. Neurosci.    211:4752-4760.-   37. Pittman, R. N., S. Wang, A. J. DiBenedetto, and J. C.    Mills. 1993. A system for characterizing cellular and molecular    events in programmed neuronal cell death. J. Neurosci. 13:3669-3680.-   38. Putcha, G. V., M. Deshmukh, and E. M. Johnson, Jr. 1999. BAX    translocation is a critical event in neuronal apoptosis regulation    by neuroprotectants, BCL-2, and caspases. J. Neurosci. 19:7476-7485.-   39. Putcha, G. V., C. A. Harris, K. L. Moulder, R. M. Easton, C. B.    Thompson, and E. M. Johnson, Jr. 2002. Intrinsic and extrinsic    pathway signaling during neuronal apoptosis: lessons from the    analysis of mutant mice. J. Cell Biol 157:441-453.-   40. Rabizadeh, S., J. Oh, L. T. Zhong, J. Yang, C. M. Bitler, L. L.    Butcher, and D. E. Bredesen. 1993. Induction of apoptosis by the    low-affinity NGF receptor. Science 261:345-348.-   41. Read, S. H., B. C. Baliga, P. G. Ekert, D. L. Vaux, and S.    Kumar. 2002. A novel Apaf-1-independent putative caspase-2    activation complex. J. Cell Biol. 159:739-745.-   42. Sawada, M., P. Hayes, and S. Matsuyama. 2003a. Cytoprotective    membrane-permeable peptides designed from the BAX-binding domain of    Ku70. Nat. Cell Biol. 5:352-357.-   43. Sawada, M., W. Sun, P. Hayes, K. Leskov, D. A. Boothman, and S.    Matsuyama. 2003b. Ku70 suppresses the apoptotic translocation of Bax    to mitochondria. Nat. Cell Biol. 5:320-329.-   44. Scorrano, L., M. Ashiya, K. Buttle, S. Weiler, S. A.    Oakes, C. A. Mannella, and S. J. Korsmeyer. 2002. A distinct pathway    remodels mitochondrial cristae and mobilizes cytochrome c during    apoptosis. Dev. Cell 2:55-67.-   45. Sperandio, S., I. de Belle, and D. E. Bredesen. 2000. An    alternative, nonapoptotic form of programmed cell death. PNAS    97:14376-14381.-   46. Strasser, A. and K. Newton. 1999. FADD/MORT1, a signal    transducer that can promote cell death or cell growth. Int. J.    Biochem. Cell Biol. 31:533-537.-   47. Sun, Y. F., L. Y. Yu, M. Saarma, T. Timmusk, and U.    Arumäe. 2001. Neuron-specific Bcl-2 homology 3 domain-only splice    variant of Bak is anti-apoptotic in neurons, but pro-apoptotic in    non-neuronal cells. J. Biol. Chem. 276:16240-16247.-   48. Thibert, C., M. A. Teillet, F. Lapointe, L. Mazelin, N. M. Le    Douarin, and P. Mehlen. 2003. Inhibition of neuroepithelial    patched-induced apoptosis by sonic hedgehog. Science 301:843-846.-   49. Tolkovsky, A. M., L. Xue, G. C. Fletcher, and V.    Borutaite. 2002. Mitochondrial disappearance from cells: a clue to    the role of autophagy in programmed cell death and disease?    Biochimie 84:233-240.-   50. Troy, C. M., S. A. Rabacchi, J. B. Hohl, J. M. Angelastro, L. A.    Greene, and M. L. Shelanski. 2001. Death in the balance: alternative    participation of the caspase-2 and -9 pathways in neuronal death    induced by nerve growth factor deprivation. J. Neurosci.    21:5007-5016.-   51. Vincenz, C. 2001. Death receptors and apoptosis. Deadly    signaling and evasive tactics. Cardiol. Clin. 19:31-43.-   52. Virdee, K., A. J. Bannister, S. P. Hunt, and A. M.    Tolkovsky. 1997. Comparison between the timing of JNK activation,    c-Jun phosphorylation, and onset of death commitment in sympathetic    neurones. J. Neurochem. 69:550-561.-   53. Xue, L., G. C. Fletcher, and A. M. Tolkovsky. 1999. Autophagy is    activated by apoptotic signalling in sympathetic neurons: an    alternative mechanism of death execution. Mol. Cell Neurosci.    14:180-198.-   54. Yaginuma, H., N. Shiraiwa, T. Shimada, K. Nishiyama, J. Hong, S.    Wang, T. Momoi, Y. Uchiyama, and R. W. Oppenheim. 2001. Caspase    activity is involved in, but is dispensable for, early motoneuron    death in the chick embryo cervical spinal cord. Mol. Cell Neurosci.    18:168-182.-   55. Yu, L., L. Korhonen, R. Martinez, E. Jokitalo, Y. Chen, U.    Arumäe, and D. Lindholm. 2003. Regulation of sympathetic neuron and    neuroblastoma cell death by XIAP and its association with    proteasomes in neural cells. Molecular and Cellular Neuroscience    22:308-318.-   56. Zaidi, A. U., C. D'Sa-Eipper, J. Brenner, K. Kuida, T. S.    Zheng, R. A. Flavell, P. Rakic, and K. A. Roth. 2001.    Bcl-x_(L)-caspase-9 interactions in the developing nervous system:    evidence for multiple death pathways. J. Neurosci. 21:169-175.-   57. Zimmermann, K. C., C. Bonzon, and D. R. Green. 2001. The    machinery of programmed cell death. Pharmacol. Ther. 92:57-70.

1. A method of screening caspase-2 and caspase-7-dependent cell deathpathway modulators of neuronal or non-neuronal cells in a GDNF familygrowth factor-dependent cell culture system, comprising the steps ofremoving the GDNF family growth factor from the culture system;introducing a candidate modulator agent into the culture system; anddetermining the activity of at least caspase-2 and caspase-7 in acultured cell.
 2. The method according to claim 1, wherein said GDNFfamily growth factor is selected from the group consisting of GDNF(glial cell line-derived neurotrophic factor), NRTN (neurturin), ARTN(artemin) and PSPN (persephin).
 3. The method according to claim 1,wherein said neuronal cells are selected from the group consisting ofcervical ganglion neurons, dorsal root ganglion cells, nodose ganglionneurons, spinal motoneurons, midbrain dopaminergic neurons, centralnoradrenergic neurons and enteric neurons.
 4. The method according toclaim 1, wherein said non-neuronal cells are selected from the groupconsisting of chromaffin cells of the adrenal medulla, cells ofembryonic kidney, differentiating spermatogonia and T-cells of thethyroid gland.
 5. The method according to claim 1, wherein saidcandidate modulator agent is from a combinatorial library selected fromthe group consisting of: biological library; peptoid library, spatiallyaddressable parallel solid phase library, solution phase library;synthetic library requiring deconvolution; “one-bead, one-compound”library; and synthetic library using affinity chromatography selection.6. The method according to claim 5, wherein said combinatorial libraryis peptide library, non-peptide oligomer library or small moleculelibrary.
 7. The method according to claim 1, wherein the determining ofthe ability of the candidate agent to modulate caspase-2 and/orcaspase-7 activity is accomplished by monitoring cell death, cellgrowth, cell attachment, neurite outgrowth, and/or cell chemotaxis. 8.The method according to claim 1, wherein the determining of the abilityof the candidate agent to modulate caspase-2 and/or caspase-7 activityis accomplished by Western blot, immunohistochemical staining using anticaspase-2 or caspase-7 antibodies, and/or fluorometric assays.
 9. Themethod according to claim 8, wherein said fluorometric assay is aFRET-assay.
 10. The method according to claim 1, wherein the determiningof the ability of the candidate agent to modulate caspase-2 and/orcaspase-7 activity is accomplished by detecting catalytic or enzymaticactivity of caspase-2 and/or caspase-7.
 11. The method according toclaim 1, wherein the determining of the ability of the candidate agentto modulate caspase-2 and/or caspase-7 activity is accomplished bydetecting a target-regulated cellular response selected from the groupconsisting of cell attachment, cell adhesion, cell growth, cell deathand cell migration.
 12. The method according to claim 1, wherein thedetermining of the ability of the candidate agent to modulate caspase-2and/or caspase-7 activity is accomplished by detecting the amount ofcaspase-2 and/or caspase-7 mRNA or protein in a cultured cell in thepresence of the candidate compound and comparing the detected amount tothe amount of caspase-2 and/or caspase-7 mRNA or protein in the culturedcell in the absence of the candidate compound.
 13. The method accordingto claim 12, wherein the amount of caspase-2 and/or caspase-7 mRNA orprotein is detected to be higher in the presence of the candidatecompound than in its absence, the candidate compound is identified as astimulator of caspase-2 and/or caspase-7 activity.
 14. The methodaccording to claim 12, wherein the amount of caspase-2 and/or caspase-7mRNA or protein is detected to be lower in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of caspase-2 and/or caspase-7 activity.
 15. The methodaccording to claim 1, wherein the ability of the candidate agent tomodulate cell death in a GDNF family growth factor-dependent cellculture system is confirmed by the detection of the presence of activityof caspase-2 and/or caspase-7 and the absence of activity of Bax,caspase-3, caspase-8, and/or caspase-9.
 16. A caspase-2 and/or caspase-7modulator detected by the method according to claim
 1. 17. Apharmaceutical composition comprising the caspase-2 and/or caspase-7modulator according to claim
 16. 18. The pharmaceutical compositionaccording to claim 17 for the treatment of a disease selected from thegroup consisting of CNS lesions, gliosis, Parkinson's disease,Alzheimer's disease, neuronal degeneration, enteric diseases, kidneydisease, immunogical diseases, and diseases of chromaffin cells.
 19. Thepharmaceutical composition according to claim 17 for treating growthfactor deprivation of cells due to a injury.
 20. A method of treatmentcomprising administering to the subject in need of the treatment atherapeutically effective amount of the composition according to claim16.
 21. The method according to claim 20, wherein said subject suffersfrom any one of the conditions defined in claim 18 or
 19. 22. A methodof screening cell death modulators of neuronal or non-neuronal cells ina GDNF family growth factor-dependent cell culture system, comprisingthe steps of removing the GDNF family growth factor from the culturesystem; introducing a candidate modulator agent into the culture system;and monitoring cell death in the cell culture.